[Not Applicable]
This invention relates assay for agents that modulate (e.g. upregulate, downregulate or completely inhibit) microtubule depolymerizing or microtubule severing proteins. Such agents will have profound effects on progression of the cell cycle and act as potent anti-mitotic agents.
The cytoskeleton constitutes a large family of proteins that are involved in many critical processes of biology, such as chromosome and cell division, cell motility and intracellular transport (Vale and Kreis, (1993) Guidebook to the Cytoskeletal and Motor Proteins New York: Oxford University Press; Alberts et al., (1994) Molecular Biology of the Cell, 788-858). Cytoskeletal proteins are found in all cells and are involved in the pathogenesis of a large range of clinical diseases. The cytoskeleton includes a collection of polymer proteins, microtubules, actin, intermediate filaments, and septins, as well as a wide variety of proteins that bind to these polymers (polymer-interacting proteins). Some of the polymer-interacting proteins are molecular motors (myosins, kinesins, dyneins) (Goldstein (1993) Ann. Rev. Genetics 27: 319-351; Mooseker and Cheney (1995) Annu. Rev. Cell Biol. 11: 633-675) that are essential for transporting material within cells (e.g., chromosomal movement during metaphase), for muscle contraction, and for cell migration. Other groups of proteins (e.g., vinculin, talin and alpha-actinin) link different filaments, connect the cytoskeleton to the plasma membrane, control the assembly and disassembly of the cytoskeletal polymers, and moderate the organization of the polymers within cells.
Given the central role of the cytoskeleton in cell division, cell migration, inflammation, and fungal/parasitic life cycles, it is a fertile system for drug discovery. Although much is known about the molecular and structural properties of cytoskeletal components, relatively little is known about how to efficiently manipulate cytoskeletal structure and function. Such manipulation requires the discovery and development of specific compounds that can predictably and safely alter cytoskeletal structure and function. However, at present, drug targets in the cytoskeleton have been relatively untapped. Extensive work has been directed towards drugs that interact with the cytoskeletal polymers themselves (e.g., taxol and vincristine), and towards motility assays (Turner et al. (1996) Anal. Biochem. 242 (1): 20-5; Gittes et al. (1996) Biophys. J 70 (1): 418-29; Shirakawa et al. (1995) J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J. 68: 2444-53; and Winkelmann et al. (1995) Biophys. J 68: 72S). Virtually no effort has been directed to finding drugs that target the cytoskeletal proteins that bind to the different filaments, which might be more specific targets with fewer unwanted side effects.
This invention pertains to the discovery that proteins (e.g. motor proteins) that either depolymerize or sever microtubules, provide good targets for modulators of such activity. Without being bound by a particular theory, it is believed that microtubule depolymerizing or severing activity is critical for normal formation and/or function of the mitotic spindle. Thus, agents that modulate (e.g., upregulate, downregulate, or completely inhibit) depolymerization or severing activity are expected to have a significant activity on progression of the cell cycle (e.g. acting as potent anti-mitotic agents).
This invention thus provides, in one embodiment, assays for identifying an agent that modulates microtubule depolymerization. The assays involve contacting a polymerized microtubule with a microtubule severing protein or a microtubule depolymerizing protein in the presence of an ATP or a GTP and the xe2x80x9ctestxe2x80x9d agent; and detecting the formation of tubulin monomers, dimers, or oligomers. The microtubule can be labeled with any of a variety of labels, however in a preferred embodiment, it is labeled with DAPI. The formation of tubulin monomers, dimers, or oligomers can be detected by any of a wide variety of methods including, but not limited to changes in DAPI fluorescence, fluorescent resonance energy transfer (FRET), centrifugation, and the like. The microtubules are preferably microtubules that are either naturally stable (e.g., axonemal microtubules) or microtubules that have been stabilized (e.g., by contact with an agent such as paclitaxel, a paclitaxel analogue, or a non-hydrolyzable nucleotide GTP analogue such as guanylyl-(xcex1,xcex2)-methylene diphosphate (GMPCPP)).
The assays can be run in solution or in solid phase (i.e. where one or more assay components are attached to a solid surface. In one embodiment, of solid-phase assays, the microtubule is attached to the surface e.g., by direct binding or by binding with an agent such as an inactivated microtubule motor protein, an avidin-biotin linkage, an anti-tubulin antibody, a microtubule binding protein (MAP), or a polylysine. The microtubule severing protein or microtubule depolymerizing protein is preferably a katanin, a p60 subunit of a katanin, an XKCM1, or an OP18 polypeptide. In a particularly preferred embodiment, the microtubule severing protein is a katanin or a p60 subunit of a katanin as described herein.
It was also a discovery of this invention that the katanin p60 subunit exhibits both the ATPase and microtubule severing activity observed in katanin. The p60 subunit thus provides a good target for screening for potential therapeutic lead compounds. Thus, in another embodiment, this invention provides methods screening for (identifying) a therapeutic lead compound that modulates depolymerization or severing of a microtubule system. The methods involve providing an assay mixture comprising a katanin p60 subunit and a microtubule, contacting the assay mixture with a test compound to be screened for the ability to inhibit or enhance the microtubule-severing or ATPase activity of the p60 subunit; and detecting specific binding of the test compound to said p60 subunit or a change in the ATPase activity of the p60 subunit. The detecting can be by any of a wide variety of methods including, but not limited to detecting ATPase activity using malachite green as a detection reagent. Binding activity can be easily detected in binding assays in which the p60 subunit is labeled and said test agent is attached to a solid support or conversely, the test agent is labeled and the p60 subunit is attached to a solid support. In a preferred embodiment, the ATPase assays are performed in the presence of stabilized microtubules.
The assay methods of this invention are also amendable to high throughput screening. Thus, in one embodiment, any of the methods described herein is performed in an array where said array comprises a multiplicity of reaction mixtures, each reaction mixture comprising a distinct and distinguishable domain of said array, and wherein the assay steps are performed in each reaction mixture. The array can take a number of formats, however, in one preferred format, the array comprises a microtitre plate, preferably a microtitre plate comprising at least 48 and more preferably at least 96 reaction mixtures. The test agent can be one of a plurality of agents and each reaction mixture can comprise one agent of the plurality of agents.
In addition, this invention provides for polypeptides having microtubule severing activity. The polypeptides comprise an isolated p60 subunit of a katanin, where the p60 subunit is encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid that encodes the katanin p60 amino acid sequence (SEQ ID NO:1). In a particularly preferred embodiment, the polypeptide is the polypeptide of SEQ ID NO:1 or the polypeptide of SEQ ID NO:1 having conservative substitutions. The polypeptide can comprise at least 8 contiguous amino acids from a polypeptide sequence encoded by a nucleic acid as set forth in SEQ ID NO:1, where the polypeptide, when presented as an antigen, elicits the production of an antibody that specifically binds to a polypeptide sequence encoded by a nucleic acid as set forth in SEQ ID NO: 1; and the polypeptide does not bind to antisera raised against a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO:1, that has been fully immunosorbed with a polypeptide encoded by a nucleic acid sequence as set forth in SEQ ID NO: 1. In a most preferred embodiment, the polypeptide is polypeptide of SEQ ID NO:1.
This invention also provides an isolated nucleic acid that encodes a katanin p60 subunit having microtubule severing activity. The nucleic acid preferably comprises a nucleic acid that specifically hybridizes with a nucleic acid that encodes the polypeptide of SEQ ID NO:1 under stringent conditions. The nucleic acid preferably encodes a polypeptide of SEQ ID NO:1 or conservative substitutions thereof. The katanin p60 encoding nucleic acid can be operably linked to a promoter (e.g. a baculovirus promoter) and may be present in a vector.
In another embodiment, this invention provides methods of screening for an agent that alters microtubule polymerization, or depolymerization, or severing. The methods involve providing labeled tubulin; contacting the labeled tubulin with the test agent to produce contacted tubulin; and comparing the fluorescence intensity or pattern of the contacted tubulin with the fluorescence intensity or pattern of labeled tubulin that is not contacted with the test agent where a difference in fluorescence pattern or intensity between the contacted and the not contacted tubulin indicates that the agent alters microtubule polymerization, or depolymerization, or severing. In particularly preferred embodiments, the labeled tubulin is in the form of tubulin monomers, tubulin dimers, tubulin oligomers, or a microtubule. In some embodiments, the microtubule is attached to a solid surface (e.g., by binding with an agent selected from the group consisting of an inactivated microtubule motor protein, an avidin-biotin linkage, an anti-tubulin antibody, a microtubule binding protein (MAP), a polyarginine, a polyhistidine, and a polylysine). Preferred labels include DAPI, ANS, Bis-ANS, ruthenium red, cresol violet, and DCVJ, with DAPI being most preferred. In some embodiments, the xe2x80x9ccontactingxe2x80x9d step can further comprise contacting the tubulin with a microtubule depolymerizing protein or a microtubule severing protein. Preferred microtubule severing or a microtubule depolymerizing proteins include, but are not limited to katanin, a p60 subunit of a katanin, an XKCM1, and a OP18 polypeptide. A preferred p60 subunit of a katanin is a polypeptide of SEQ ID NO: 1. The method can further involve listing the agents that alter microtubule polymerization, depolymerization, or severing into a database of therapeutic lead compounds that act on the cytoskeletal system. This method can be performed in various array embodiments as described herein.
This invention also provides kits for practice of any of the methods described herein. In one embodiment, the kits comprise one or more containers containing an isolated microtubule severing protein or a microtubule depolymerizing protein. The kit can further comprise a polymerized microtubule labeled with DAPI. The microtubule can be stabilized by contact with paclitaxel or a paclitaxel derivative. The microtubule can also optionally be attached to a solid surface (e.g., by binding with an inactivated motor protein). The microtubule severing protein or microtubule depolymerizing protein is preferably selected from the group consisting of a katanin, a p60 subunit of a katanin, an XKCM1, and a OP18 polypeptide. In a particularly preferred embodiment, the microtubule severing protein is a katanin or a p60 subunit of a katanin.
The term xe2x80x9cnucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol Chem. 260: 2605-2608; Cassol et al. (1992); and Rossolini et al, (1994) Mol Cell Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
The terms xe2x80x9cpolypeptide,xe2x80x9d xe2x80x9cpeptide,xe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are preferably in the natural xe2x80x9cLxe2x80x9d isomeric form. However, residues in the xe2x80x9cDxe2x80x9d isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. In addition, the amino acids, in addition to the 20 xe2x80x9cstandardxe2x80x9d amino acids, include modified and unusual amino acids, which include, but are not limited to those listed in 37 CFR xc2xa71.822(b)(4). Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.
The term xe2x80x9cconservative substitutionxe2x80x9d is used in reference to proteins or peptides to reflect amino acid substitutions that do not substantially alter the activity (specificity or binding affinity) of the molecule. Typically conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g. charge or hydrophobicity). The following six groups each contain amino acids that are typical conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
The terms xe2x80x9cisolatedxe2x80x9d and xe2x80x9cbiologically purexe2x80x9d refer to material which is substantially or essentially free from components which normally accompany it as found in its native state. However, the term xe2x80x9cisolatedxe2x80x9d is not intended to refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu.
The terms xe2x80x9cidentical,xe2x80x9d percent xe2x80x9cidentity,xe2x80x9d and percent xe2x80x9chomologyxe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95% or even at least 98% amino acid residue identity across a window of at least 30 nucleotides, preferably across a window of at least 40 nucleotides, more preferably across a window of at least 80 nucleotides, and most preferably across a window of at least 100 nucleotides, 150 nucleotides, 200 nucleotides or greater, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (See generally, Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J Mol. Evol. 3 5:3 51-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.nebi.rilm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length xe2x80x9cWxe2x80x9d in the query sequence, which either match or satisfy some positive-valued threshold score xe2x80x9cTxe2x80x9d when aligned with a word of the same length in a database sequence. xe2x80x9cTxe2x80x9d is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity xe2x80x9cXxe2x80x9d from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters xe2x80x9cW,xe2x80x9d xe2x80x9cT,xe2x80x9d and xe2x80x9cXxe2x80x9d determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (xe2x80x9cWxe2x80x9d) of 11, the BLOSUM62 scoring matrix (See Henikoff and Henikoff (1989) Proc. Natl. Acad Sci USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (See, e.g., Karlin and Altschul (1993) Proc. Natl. Acad Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
The phrases xe2x80x9chybridizing specifically to,xe2x80x9d xe2x80x9cspecific hybridization,xe2x80x9d and xe2x80x9cselectively hybridize to,xe2x80x9d refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
The term xe2x80x9cstringent conditionsxe2x80x9d refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. Stringent hybridization and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 Overview of Principles of Hybridization and the Strategy of Nucleic Acid Probe Assays, Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42xc2x0 C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (See, Sambrook et al. (1989) Molecular Cloningxe2x80x94A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex (e.g., of more than 100 nucleotides), is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example low stringency wash for a duplex (e.g., of more than 100 nucleotides), is 4-6x SSC at 40xc2x0 C. for 15 minutes. In general, a signal to noise ratio of 2xc3x97 (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The terms xe2x80x9ckataninxe2x80x9d or xe2x80x9ckatanin p60 subunitxe2x80x9d refer to katanin and the katanin p60 subunit as described herein, in the references cited and in the sequence listings. The terms also include proteins having substantial amino acid sequence identity with katanin or the katanin p60 subunit sequences provided herein that exhibit ATPase and microtubule severing activity.
The terms xe2x80x9ctaxolxe2x80x9d and xe2x80x9ctaxol derivatives or analogues refer to the drug taxol known generically as paclitaxel (NSC number: 125973). Paclitaxel (taxol) derivatives and analogues show similar microtubule-stabilizing activity. Preferred derivatives include taxotere and others.
Depolymerized microtubule components are defined so as to include the products of microtubule depolymerization or severing, and include tubulin monomers, dimers and oligomers.
The term xe2x80x9ctest agentxe2x80x9d refers to an agent that is to be screened in one or more of the assays described herein. The agent can be virtually any chemical compound. It can exist as a single isolated compound or can be a member of a chemical. (e.g., combinatorial) library. In a particularly preferred embodiment, the test agent will be a small organic molecule.
The term small organic molecules refers to molecules of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
The terms xe2x80x9clabelxe2x80x9d or xe2x80x9cdetectable labelxe2x80x9d are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads(trademark)), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.